Oxford-Nanopore eBook - 6

WORKFLOW: STRUCTURAL VARIATION

Resolving structural variants with long-read
nanopore sequencing
Structural variants (SVs), defined as variants spanning 50 bp or more, account for ten times as many variant bases as
single nucleotide polymorphisms (SNPs) in the human genome1. With known causative effects in an extensive range
of both normal and aberrant phenotypes, the need to comprehensively characterise SVs is becoming increasingly
clear. With Oxford Nanopore, long-read sequencing of native DNA
greatly improves the accuracy of detection of even the largest of SVs,
including those regions inaccessible to other technologies2.

Here, we present a simple workflow for an effective
whole-genome SV survey from a human blood
sample, using the PromethION™ sequencing device.

Selecting a suitable extraction
method is often a trade-off
between input requirements,
expected fragment lengths, lab
experience and hands-on time.
To maximise the volume of data
at long read lengths, we
recommend the QIAGEN
Gentra Puregene Blood Kit.

Find more extraction protocol recommendations for your sample
type, plus guidance on DNA storage and contaminants:
community.nanoporetech.com/extraction_methods
Circulomics
QIAGEN Gentra Puregene

Bases sequenced

EXTRACTION:
obtaining high molecular weight DNA

QIAamp DNA Blood Midi Kit
QIAGEN Genomic-tip

0

25

50

75

100

Read length (kb)

LIBRARY PREPARATION:
selecting a kit

Find out more about size selection methods for
long-read sequencing:
community.nanoporetech.com/extraction_methods

There is no upper read length limit in nanopore sequencing, with
reads routinely spanning tens or hundreds of kilobases and the
current record spanning over 2 megabases. Fragmentation is
optional: unfragmented DNA offers a simple workflow, but shearing
and size selection can improve read N50. Internal testing has yielded
good results from the Circulomics Short Read Eliminator Kit for
size selection, and the Diagenode Megaruptor 3 for shearing.

High molecular weight gDNA

Optional fragmentation
or size selection

End-prep and nick repair
60 min

A

p

p

A

Ligation of sequencing
adapters

T

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To prepare gDNA for sequencing, we recommend the Ligation
Sequencing Kit (SQK-LSK109), providing the greatest
throughput and control over read lengths. Gentle pipetting with
wide-bore tips can also help preserve long DNA strands.

Find out more about sample prep, including rapid and multiplexing options: nanoporetech.com/products/kits
http://community.nanoporetech.com/extraction_methods http://community.nanoporetech.com/extraction_methods http://www.nanoporetech.com/products/kits
Oxford-Nanopore eBook

Table of Contents for the Digital Edition of Oxford-Nanopore eBook
